DNA detection is described in European Patent Application 381,501 using a method wherein PCR amplification of miniscule amounts of nucleic acid material, and detection of the amplified material can all occur in a single pouch that keeps the amplified material from escaping. Six temporarily-sealed blisters, also called compartments, are provided along with passageways connecting them to a detection site in a detection compartment. The blisters provide, in order, a PCR reaction compartment; a first wash compartment; an enzyme-labeling compartment containing, e.g., streptavidin horseradish peroxidase (hereinafter SA-HRP); a second wash compartment; a compartment containing signalling material responsive to the enzyme; and a stop solution compartment. Each of these is caused to empty into the detection compartment in the order indicated, where a detection site is used to capture the amplified nucleic acid material and to generate a detectable signal.
The use of the two wash compartments to provide two wash steps is consistent with all conventional approaches to detecting nucleic acid material. For example, Vol. 30 of J. Clin. Microbiol, 845-853 (April, 1992) describes a process used by Roche (p. 846-847) as being one in which, following hybridization of biotinylated product to the solid wall surface, "we washed the plate 4 times with wash Buffer I to remove any unhybridized product". These four washes correspond to the first wash step of the first wash blister of the pouch of European Patent Application 381,501, since there also, any DNA or nucleic acid material "unhybridized" to the detection sites is washed off. Thereafter, the Roche procedure incubates "at 37.degree. C. for 15 minutes with an avidin-horseradish peroxidase conjugate", which of course corresponds to the emptying of the enzyme blister of the EPA pouch for the very same purpose. Thereafter, the Roche procedure" again washed the plate four times" "to remove unbound conjugate." This, of course, corresponds to the second wash step provided by the second wash blister disposed between the enzyme blister and the signalling material blister in the pouch of EPA 381,501.
Such procedures, with all the washes, although quite workable, are time consuming and therefore expensive. Further, the washes introduce complications into the manufacture of the pouch. However, they have been considered essential in order to eliminate "nonspecific signal," that is, signal that occurs because of either the presence of unbound nucleic acid material that is NOT the target, and/or unbound SA-HRP that should not be present because the target nucleic acid material is not present.
Thus, there has been a need prior to this invention to come up with a detection sequence that eliminates at least one, and preferably both, of the wash steps and wash blisters heretofore needed, without causing so much noise in the detection as to make the signal unreliable.